严重发热伴血小板减少综合征病毒核蛋白核酸疫苗的构建及免疫原性研究
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国家重大科技专项(2013ZX10002005-002-005);江苏省医学创新团队与领军人才资助(LJ201121);江苏省科技支撑计划资助项目(BE2012770);江苏高校优势学科建设工程资助(JX10231801)


Construction of nucleic acid vaccine plasmid of severe fever with thrombocytopenia syndrome virus nucleoprotein and immunogenicity study
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    摘要:

    目的:构建SFTSV的核蛋白核酸疫苗并探讨其免疫原性-方法:采用PCR方法扩增SFTSV核蛋白基因,并用引物引入限制性内切酶酶切位点,克隆入载体pJW4303-经酶切和测序鉴定正确的质粒,用PEI瞬时转染HEK293T细胞,用免疫印迹法鉴定核蛋白的表达-同时以质粒pJW4303作为阴性对照,免疫BALB/c小鼠,酶联免疫法验证其免疫原性-结果:成功构建质粒pJW4303-N,经Western blot证实SFTSV核蛋白能够在HEK293T细胞中表达-ELISA检测免疫后小鼠血清特异性IgG抗体及其亚型发现,IgG抗体及其亚型较免疫前明显升高,亚型中以IgG2a升高为主-结论:SFTSV核蛋白核酸疫苗pJW4303-N具有良好的免疫原性,为进一步研究SFTSV核蛋白奠定了基础-

    Abstract:

    Objective:To construct nucleic acid vaccine plasmid encoding severe fever with thrombocytopenia syndrome virus(SFTSV) nucleoprotein and to study its immunogenicity. Methods: SFTSV nucleoprotein gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the expression vector pJW4303 to construct nucleic acid vaccine. The recombinant plasmid pJW4303-N was identified by sequencing and then transiently transformed into HEK293T cells to measure the nucleoprotein expression by Western blot. We immuned the BALB/c mice with pJW4303-N and used the blank vector pJW4303 as the control. The immunogenicity was detected by enzyme-linked immunosorbent method. Results: The recombinant SFTSV nucleoprotein expression vector pJW4303-N was successfully constructed and expressed in supernatants and cell lysates of HEK293T cells in vitro. Specific IgG antibodies and its subtype in mice were found by ELISA after immunization. IgG titer and its subsets in the serum of the BALB/c mice were significantly increased than those of the controls. IgG2a was significantly higher than IgG1. Conclusion: The nucleic acid vaccine of SFTSV nucleoprotein (pJW4303-N) has good immunogenicity. This research is the foundation for the further study of SFTSV nucleoprotein.

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周宜庆,金 柯,徐菱遥,李 军.严重发热伴血小板减少综合征病毒核蛋白核酸疫苗的构建及免疫原性研究[J].南京医科大学学报(自然科学版),2015,(7):928-932

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  • 收稿日期:2015-03-21
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  • 在线发布日期: 2015-07-12
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